U.S. patent application number 12/306495 was filed with the patent office on 2010-04-15 for porphyromonas gingivalis polypeptides useful in the prevention of periodontal disease.
This patent application is currently assigned to ORAL HEALTH AUSTRALIA PTY LTD. Invention is credited to Ching Seng Ang, Stuart Geoffrey Dashper, Eric Charles Reynolds, Paul David Veith.
Application Number | 20100092471 12/306495 |
Document ID | / |
Family ID | 38845037 |
Filed Date | 2010-04-15 |
United States Patent
Application |
20100092471 |
Kind Code |
A1 |
Dashper; Stuart Geoffrey ;
et al. |
April 15, 2010 |
Porphyromonas Gingivalis Polypeptides Useful in the Prevention of
Periodontal Disease
Abstract
The invention is directed to vaccine compositions and methods
based on P. gingivalis proteins identified to be regulated by haem
availability that can be used in the prevention and treatment of
periodontal disease. In particular, two specific internalin-like P.
gingivalis proteins, namely PG0350 and PG1374 involved in the
internalization of P. gingivalis by host cells, the hypothetical
protein, PG1019 purported to be a cell surface lipoprotein and the
alkyl hydroperoxide reductase protein, PG0618 have been identified
as useful targets for the prevention and treatment of periodontal
disease.
Inventors: |
Dashper; Stuart Geoffrey;
(Brunswick East, AU) ; Ang; Ching Seng;
(Kensington, AU) ; Veith; Paul David; (Ringwood
East, AU) ; Reynolds; Eric Charles; (Balwyn,
AU) |
Correspondence
Address: |
DLA PIPER LLP (US)
4365 EXECUTIVE DRIVE, SUITE 1100
SAN DIEGO
CA
92121-2133
US
|
Assignee: |
ORAL HEALTH AUSTRALIA PTY
LTD
Carlton, Victoria
AU
|
Family ID: |
38845037 |
Appl. No.: |
12/306495 |
Filed: |
June 27, 2007 |
PCT Filed: |
June 27, 2007 |
PCT NO: |
PCT/AU2007/000890 |
371 Date: |
December 23, 2008 |
Current U.S.
Class: |
424/139.1 ;
514/1.1; 514/2.8; 514/6.9; 530/387.9 |
Current CPC
Class: |
G01N 2333/195 20130101;
A61K 39/00 20130101; A61P 1/02 20180101; G01N 2800/18 20130101;
C07K 14/195 20130101; A61K 38/164 20130101; A61K 39/0216
20130101 |
Class at
Publication: |
424/139.1 ;
514/15; 514/14; 514/13; 514/12; 530/387.9 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 38/08 20060101 A61K038/08; A61K 38/10 20060101
A61K038/10; A61P 43/00 20060101 A61P043/00; A61K 38/16 20060101
A61K038/16; C07K 16/00 20060101 C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 27, 2006 |
AU |
2006903425 |
Claims
1. (canceled)
2. A composition according to claim 4 in which the amino acid
sequence is 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or
SEQ ID NO:4.
3. A composition according to claim 4 in which the amino acid
sequence comprises a contiguous sequence of at least 20, 30, 40,
50, 60, 70, 80, 90 or 100 amino acids which is identical to a
contiguous amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ
ID NO:3 or SEQ ID NO:4.
4. A composition for use in raising an immune response directed
against P. gingivalis in a subject, the composition comprising an
effective amount of at least one amino acid sequence comprising at
least 10 amino acids identical to a contiguous amino acid sequence
of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 and a
pharmaceutically acceptable carrier.
5. A method of preventing or treating a subject for periodontal
disease comprising administering to the subject the composition
according to claim 4.
6. (canceled)
7. A method according to claim 5 in which the subject is selected
from the group consisting of humans, sheep, cattle, horses, bovine,
pigs, poultry, dogs and cats.
8. A method according to claim 7 in which the subject is a
human.
9. An antibody which binds specifically to a polypeptide according
to claim 4.
10. An antibody of claim 9 which is a polyclonal antibody.
11. An antibody of claim 9 which is a monoclonal antibody.
12. A composition useful in the prevention or treatment of
periodontal disease, the composition comprising an antibody
according to claim 9 and a pharmaceutically acceptable carrier.
13-20. (canceled)
21. A composition according to claim 4, wherein the amino acid
sequence is the PG0350 protein having the amino acid sequence of
SEQ ID NO:1.
22. A composition according to claim 4, wherein the amino acid
sequence is the PG1374 protein having the amino acid sequence of
SEQ ID NO:2.
23. A composition according to claim 4, wherein the amino acid
sequence is the PG1019 protein having the amino acid sequence of
SEQ ID NO:3.
24. A composition according to claim 4, wherein the amino acid
sequence is the PG0618 protein having the amino acid sequence of
SEQ ID NO:4.
25. A method according to claim 5, wherein the composition
comprises at least one amino acid sequence comprising at least 10
amino acids identical to a contiguous amino acid sequence of the
PG0350 protein having the amino acid sequence of SEQ ID NO:1.
26. A method according to claim 5, wherein the composition
comprises at least one amino acid sequence comprising at least 10
amino acids identical to a contiguous amino acid sequence of the
PG1374 protein having the amino acid sequence of SEQ ID NO:2.
27. A method according to claim 5, wherein the composition
comprises at least one amino acid sequence comprising at least 10
amino acids identical to a contiguous amino acid sequence of the
PG1019 protein having the amino acid sequence of SEQ ID NO:3.
28. A method according to claim 5, wherein the composition
comprises at least one amino acid sequence comprising at least 10
amino acids identical to a contiguous amino acid sequence of the
PG0618 protein having the amino acid sequence of SEQ ID NO:4.
29. A method according to claim 5, wherein the composition
comprises an amino acid sequence at least 85% identical to the
amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or
SEQ ID NO:4.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions and methods of
isolating Porphyromonas gingivalis (P. gingivalis) proteins useful
in the prevention of and treatment of periodontal disease. More
particularly, the invention is directed to vaccine compositions and
methods based on P. gingivalis proteins identified to be regulated
by haem availability that can be used in the prevention and
treatment of periodontal disease.
BACKGROUND OF THE INVENTION
[0002] Periodontal disease is a chronic bacterial infection that
affects the gums and bone supporting the teeth. Periodontal disease
begins when the bacteria in plaque (the sticky biofilm that
constantly forms on teeth) causes the gums to become inflamed.
Periodontal disease can affect the gingival tissue (gums);
periodontal membrane (connective tissue embedded in the cementum
and alveolar bone); cementum (mineralized connective tissue
covering the roots of the teeth); and the alveolar bone (bone
socket), Depending on the progression of the disease, there may
occur a destruction of periodontal membranes, alveolar bone loss,
and apical migration of the connective tissue attachment. Advanced
periodontal disease may result in the formation of periodontal
pockets harbouring bacterial plaque, and progressive loosening and
eventual loss of teeth. Periodontal disease includes gingivitis
that can advance to periodontitis. Chronic periodontitis is an
inflammatory disease of the supporting tissues of teeth that is
associated with specific bacteria in subgingival dental plaque. The
disease has been estimated to affect around 35% of dentate adults
and is a major cause of tooth loss in the Western world.sup.(1). P.
gingivalis, a member of the normal oral microflora of subgingival
dental plague, has been implicated as one of the major
opportunistic pathogens in the progression of this
disease.sup.(2).
[0003] P. gingivalis is a black-pigmented, asaccharolytic,
Gram-negative anaerobic, cocco-bacillus, that relies on the
fermentation of amino acids for energy production.sup.(3). Like
most bacteria, P. gingivalis has an essential growth requirement
for iron that it preferentially acquires in the form of haem, a
molecule comprised of a protoporphyrin IX ring (PPIX) with a
co-ordinated central ferrous atom.sup.(4). This utilization of haem
as an iron source may reflect the inability of P. gingivalis to
synthesize PPIX de novo.sup.(5). Haem is preferentially obtained
from haemoglobin, and is acquired through the activity of the
cell-surface Arg- and Lys-specific proteinase/adhesin
complex.sup.(4,6,7), possibly in conjunction with a TonB-linked
outer membrane receptor, HmuR.sup.(8). Unlike aerobic or
facultative bacteria that obtain iron using siderophores P.
gingivalis does not produce siderophores and lacks the ferric
reductase activity usually associated with siderophore-mediated
iron acquisition.sup.(9,10). P. gingivalis stores haem on its
surface in the form of .mu.-oxo bis-haem, which has inherent
catalase activity that helps to protect the cell from oxidative
attack.sup.(11). For P. gingivalis to be able to compete with the
large numbers and diversity of bacteria within the
micronutrient-limiting environment of the oral cavity.sup.(12) it
not only has to establish itself but also has to evade or overcome
numerous host defenses.
[0004] The initiation and progression of periodontal disease is
associated with bleeding at the site of disease, thereby providing
an elevated level of haemoglobin. Therefore in order to help
understand the mechanism by which P. gingivalis establishes and
proliferates in subgingival plaque and initiates disease it is
important to determine the changes in relative protein abundances
of P. gingivalis during the transition from micronutrient poor
(haem-limitation) to rich (haem-excess) conditions.
[0005] Although many proteins have been associated with growth
under haem-limitation.sup.(9,13), no extensive work on the P.
gingivalis proteome or the changes to the proteome during
haem-limitation has been reported.
[0006] In developing compositions which would be useful in the
prevention and treatment of periodontal disease it is desirable to
identify agents that interfere and prevent the initial stages of
the disease process.
[0007] The present inventors have now developed methods for
identifying specific P. gingivalis proteins regulated by haem
availability that can be used as suitable targets for the
prevention and treatment of periodontal disease.
SUMMARY OF THE INVENTION
[0008] The present inventors have successfully developed methods of
identifying P. gingivalis proteins regulated by haem availability
that are responsible for P. gingivalis metabolism, virulence and
invasion of host cells. In particular, two specific internalin-like
P. gingivalis proteins, namely PG0350, PG1374 involved in the
internalization of P. gingivalis by host cells, a hypothetical
protein, PG1019 purported to be a cell surface lipoprotein and an
alkyl hydroperoxide reductase protein, PG0618 have been identified
as useful targets for the prevention and treatment of periodontal
disease.
[0009] A first aspect of the invention is an isolated antigenic P.
gingivalis polypeptide, the polypeptide being selected from the
group consisting of: [0010] (i) the PG0350 protein having the amino
acid sequence of SEQ ID NO:1; [0011] (ii) the PG1374 protein having
the amino acid sequence of SEQ ID NO:2; [0012] (iii) the PG1019
protein having the amino acid sequence of SEQ ID NO:3; [0013] (iv)
the PG0618 protein having the amino acid sequence of SEQ ID NO:4;
[0014] (v) an amino acid sequence at least 85% identical to the
amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or
SEQ ID NO:4; or [0015] (vi) an amino acid sequence comprising at
least 10 amino acids identical to a contiguous amino acid sequence
of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4.
[0016] A second aspect of the invention is a vaccine composition
for use in raising an immune response directed against P.
gingivalis in a subject, the composition comprising an effective
amount of at least one polypeptide of the first aspect of the
invention and a pharmaceutically acceptable carrier.
[0017] A third aspect of the invention is a method of preventing or
treating a subject for periodontal disease comprising administering
to the subject a vaccine composition according to the present
invention.
[0018] A fourth aspect of the invention is an antibody raised
against a polypeptide of the first aspect of the present invention.
Preferably, the antibody binds specifically to the polypeptides of
the present invention.
[0019] A fifth aspect of the invention is a composition useful in
the prevention or treatment of periodontal disease, the composition
comprising an antibody of the fourth aspect of the present
invention and a pharmaceutically acceptable carrier.
[0020] In a sixth aspect of the present invention there is provided
a method of identifying a P. gingivalis polypeptide involved in the
progression of periodontal disease, wherein the method comprises
the steps of:
a) determining the relative amount of a polypeptide or peptide
thereof produced by P. gingivalis grown under haem limited
conditions; and b) determining the relative amount of the
polypeptide or peptide thereof produced by P. gingivalis grown
under higher haem conditions than step a); wherein an increase in
the amount of the polypeptide or peptide fragment thereof detected
in step a) compared to step by indicates that the polypeptide is
involved in the progression of periodontal disease.
[0021] In a seventh aspect of the present invention there is
provided an interfering RNA molecule, the molecule comprising a
double stranded region of at least 19 base pairs in each Strand
wherein one of the strands of the double stranded region is
complementary to a region of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID
NO; 7 or SEQ ID NO: 8.
[0022] In an eighth aspect of the present invention there is
provided for the use of at least one polypeptide of the first
aspect of the present invention in the manufacture of a medicament
for the treatment of periodontal disease in a subject.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1 shows a diagrammatic representation of the combined
strategy used to identify proteins from P. gingivalis grown under
haem-limitation. The lysed cells were prefractionated into soluble
and insoluble fractions using ultra-centrifugation, followed by
analysis of these two fractions. The separation and analysis
procedure consists of two main methods; LCMS with gas phase
fractionation and geLCMS.
[0024] FIG. 2 shows a graph indicating the codon adaptation index
(CAI) distribution of the identified P. gingivalis proteome and the
predicted P. gingivalis genome calculated using INCA.sup.(14).
Ribosomal and tRNA synthases were defined as highly expressed
genes. Genes with less than 100 codons were excluded, bringing the
total calculated genes to 1685.
[0025] FIG. 3 shows the analysis and identification of PG0390 based
on detection of a single peptide (A) Total ion chromatogram. (B)
Mass spectrum at 54.8 min, insert showing an enlarged ICAT peptide
ion pair at 703.9 and 708.4 m/z of ratio 1:2 (L/H). (C) Product ion
spectrum for precursor 703.9 m/z. This peptide ion was identified
as having the sequence LVDLNC*FDIK (MASCOT score=40; C* denotes
ICAT modified cysteine).
[0026] FIG. 4 shows the distribution of protein abundance based on
ratio of haem-limitation over haem-excess (every one unit on the
Log 2 scale indicates a two fold change).
[0027] FIG. 5 shows binding to KB cells by P. gingivalis W50
(.diamond-solid.) and ECR312 (.box-solid.). The assay was carried
out with two biological replication (n=6). Insert shows the gating
of live KB cells based on forward and side scattering properties
(top left), five peaks representing FITC fluorescence of hound P.
gingivalis W50 to KB cells at P. gingivalis:KB cell ratios of 250
to 1250 (top right) and ECR312 (bottom right).
[0028] FIG. 6 shows the amino acid sequence of PG0350 protein
(referred to as SEQ ID NO:1 as also indicated in the sequence
listing).
[0029] FIG. 7 shows the amino acid sequence of PG1374 protein.
(referred to as SEQ ID NO:2 as also indicated in the sequence
listing).
[0030] FIG. 8 shows the amino acid sequence of PG1019 protein
(referred to as SEQ ID NO:3 as also indicated in the sequence
listing).
[0031] FIG. 9 shows the amino acid sequence of PG0618 protein
(referred to as SEQ ID NO:4 as also indicated in the sequence
listing).
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention advantageously provides the
identification of P. gingivalis proteins regulated by haem
availability as useful targets for the prevention and treatment of
periodontal disease. Preferably, the invention provides the
identification of P. gingivalis proteins upregulated by
haem-limitation as useful targets for the prevention and treatment
of periodontal disease.
[0033] In particular, the invention provides an isolated antigenic
P. gingivalis polypeptide, the polypeptide being selected from the
group consisting of: [0034] (i) the PG0350 protein having the amino
acid sequence of SEQ ID NO; 1; [0035] (ii) the PG1374 protein
having the amino acid sequence of SEQ ID NO:2; [0036] (iii) the
PG1019 protein having the amino acid sequence of SEQ ID NO:3;
[0037] (iv) the PG0618 protein having the amino acid sequence of
SEQ ID NO:4; [0038] (v) an amino acid sequence at least 85%
identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2
or SEQ ID NO:3 or SEQ ID NO:4; or [0039] (vi) an amino acid
sequence comprising at least 10 amino acids identical to a
contiguous amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ
ID NO:3 or SEQ ID NO:4.
[0040] Preferably, the isolated antigenic polypeptide is 90%, 95%,
96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of
SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4.
[0041] Preferably, the isolated antigenic polypeptide comprises an
amino acid sequence comprising at least 20, 30, 40, 50, 60, 70, 80,
90 or 100 amino acids identical to a contiguous amino acid sequence
of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4.
[0042] In a preferred embodiment, the antigenic polypeptides
comprise amino acid sequences that compose the hydrophilic,
surface-exposed regions of the PG1374 or PG0350 or PG1019 or PG0618
protein.
[0043] The terms "peptides, proteins, and polypeptides" are used
interchangeably herein. The polypeptides of the present invention
can include recombinant polypeptides such as fusion polypeptides.
Methods for the production of a fusion polypeptide are well-known
to those skilled in the art.
[0044] As will be well understood by those skilled in the art
alterations may be made to the amino acid sequences set out in the
Sequence Listings. These alterations may be deletions, insertions,
or substitutions of amino acid residues. The altered polypeptides
can be either naturally occurring (that is to say, purified or
isolated from a natural source) or synthetic (for example, by
site-directed metagenesis on the encoding DNA). It is intended that
such altered polypeptides which have at least 85%, preferably at
least 90%, 95%, 96%, 97%, 98% or 99% identity with the sequences
set out in the Sequence Listing are within the scope of the present
invention. Antibodies raised against these altered polypeptides
will also bind to the polypeptides having one of the sequences set
out in the Sequence Listings.
[0045] Whilst the concept of conservative substitution is well
understood by the person skilled in the art, for the sake of
clarity conservative substitutions are those set out below.
TABLE-US-00001 Gly, Ala, Val, Ile, Leu, Met; Asp, Glu, Ser; Asn,
Gln; Ser, Thr; Lys, Arg, His; Phe, Tyr, Trp, His; and Pro,
N.alpha.-alkalamino acids.
[0046] The practice of the invention will employ, unless otherwise
indicated, conventional techniques of chemistry, molecular biology,
microbiology, recombinant DNA, and immunology well known to those
skilled in the art. Such techniques are described and explained
throughout the literature in sources such as, Perbal, A Practical
Guide to Molecular Cloning, John Wiley and Sons (1984).sup.(15),
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbour Laboratory Press (1989).sup.(16), Brown (editor),
Essential Molecular Biology: A Practical Approach, Volumes 1 and 2,
IRL Press (1991).sup.(17), Glover & Hames (editors), DNA
Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and
1996).sup.(18), and Ausubel et al., (Editors), Current Protocols in
Molecular Biology, Greene Pub. Associates and Wiley-Interscience
(1988, including all updates until present).sup.(19). The
disclosure of these texts are incorporated herein by reference.
[0047] An "isolated polypeptide" as used herein refers to a
polypeptide that has been separated from other proteins, lipids,
and nucleic acids with which it naturally occurs or the polypeptide
or peptide may be synthetically synthesised. Preferably, the
polypeptide is also separated from substances, for example,
antibodies or gel matrix, for example, polyacrylamide, which are
used to purify it. Preferably, the polypeptide constitutes at least
10%, 20%, 50%, 70%, and 80% of dry weight of the purified
preparation. Preferably, the preparation contains a sufficient
amount of polypeptide to allow for protein sequencing (i.e. at
least 1, 10, or 100 mg).
[0048] The isolated polypeptides described herein may be purified
by standard techniques, such as column chromatography (using
various matrices which interact with the protein products, such as
ion exchange matrices, hydrophobic matrices and the like), affinity
chromatography utilizing antibodies specific for the protein or
other ligands which bind to the protein.
[0049] An "antigenic polypeptide" used herein is a moiety, such as
a polypeptide, analog or fragment thereof, that is capable of
binding to a specific antibody with sufficiently high affinity to
form a detectable antigen-antibody complex. Preferably, the
antigenic polypeptide comprises an immunogenic component that is
capable of eliciting a humoral and/or cellular immune response in a
host animal.
[0050] A "contiguous amino acid sequence" as used herein refers to
a continuous stretch of amino acids.
[0051] In determining whether or not two amino acid sequences fall
within a specified percentage limit, those skilled in the art will
be aware that it is necessary to conduct a side-by-side comparison
or multiple alignments of sequences. In such comparisons or
alignments, differences will arise in the positioning of
non-identical residues, depending upon the algorithm used to
perform the alignment. In the present context, reference to a
percentage identity or similarity between two or more amino acid
sequences shall be taken to refer to the number of identical and
similar residues respectively, between said sequences as determined
using any standard algorithm known to those skilled in the art. For
example, amino acid sequence identities or similarities may be
calculated using the GAP programme and/or aligned using the PILEUP
programme of the Computer Genetics Group, Inc., University Research
Park, Madison, Wis., United States of America.sup.(20). The GAP
programme utilizes the algorithm of Needleman and Wunsch.sup.(21)
to maximise the number of identical/similar residues and to
minimise the number and length of sequence gaps in the alignment.
Alternatively or in addition, wherein more than two amino acid
sequences are being compared, the Clustal W programme.sup.(22) is
used.
[0052] The present invention also provides a vaccine composition
for use in raising an immune response directed against P.
gingivalis in a subject, the composition comprising an effective
amount of at least one polypeptide of the first aspect of the
invention and a pharmaceutically acceptable carrier.
[0053] The vaccine composition of the present invention preferably
comprises an antigenic polypeptide that comprises at least one
antigen that can be used to confer immunity against P. gingivalis.
The subject treated by the method of the invention may be selected
from, but is not limited to, the group consisting of humans, sheep,
cattle, horses, bovine, pigs, poultry, dogs and cats. Preferably,
the subject is a human. An immune response directed against P.
gingivalis is achieved in a subject, when the subject's immune
system produces antibodies against the specific antigenic
polypeptides.
[0054] The vaccine composition is preferably administered to a
subject to induce immunity to P. gingivalis and thereby prevent
periodontal disease. The term "effective amount" as used herein
means a dose sufficient to elicit an immune response against P.
gingivalis. This will vary depending on the subject and the level
of P. gingivalis infection and ultimately will be decided by the
attending scientist, physician or veterinarian.
[0055] The vaccine composition of the present invention comprises a
suitable pharmaceutically-acceptable carrier, such as diluent
and/or adjuvant suitable for administration to a human or animal
subject. The vaccine preferably comprises a suitable adjuvant for
delivery orally by nasal spray, or by injection to produce a
specific immune response against P. gingivalis. A vaccine of the
present invention can also be based upon a recombinant nucleic acid
sequence encoding an antigenic polypeptide of the present
invention, wherein the nucleic acid sequence is incorporated into
an appropriate vector and expressed in a suitable transformed host
(e.g. E. coli, Bacillus subtilis, Saccharomyces cerevisiae, COS
cells, CHO cells and HeLa cells) containing the vector. The vaccine
can be produced using recombinant DNA methods as illustrated
herein, or can be synthesized chemically from the amino acid
sequence described in the present invention. Additionally,
according to the present invention, the antigenic polypeptides may
be used to generate P. gingivalis antisera useful for passive
immunization against periodontal disease and infections caused by
P. gingivalis.
[0056] Various adjuvants known those skilled in the art are
commonly used in conjunction with vaccine formulations. The
adjuvants aid by modulating the immune response and in attaining a
more durable and higher level of immunity using smaller amounts of
vaccine antigen or fewer doses than if the vaccine antigen were
administered alone. Examples of adjuvants include incomplete
Freunds adjuvant (IFA), Adjuvant 65 (containing peanut oil, mannide
monooleate and aluminium monostrearate), oil emulsions, Ribi
adjuvant, the pluronic polyols, polyamines, Avridine, Quil A,
saponin, MPL, QS-21, and mineral gels such as aluminium salts.
Other examples include oil in water emulsions such as SAF-1, SAF-0,
MF59, Seppic ISA720, and other particulate adjuvants such as ISCOMs
and ISCOM matrix. An extensive but exhaustive list of other
examples of adjuvants are listed in Cox and Coulter 1992.sup.(23).
In addition to the adjuvant the vaccine may include conventional
pharmaceutically acceptable carriers, excipients, fillers, buffers
or diluents as appropriate. One or more doses of the vaccine
containing adjuvant may be administered prophylactically to prevent
periodontal disease or therapeutically to treat already present
periodontal disease.
[0057] In another preferred vaccine composition the preparation is
combined with a mucosal adjuvant and administered via the oral or
nasal mute. Examples of mucosal adjuvants are cholera toxin and
heat labile E. coli toxin, the non-toxic B sub-units of these
toxins, genetic mutants of these toxins which have reduced
toxicity. Other methods which may be utilised to deliver the
antigenic polypeptides orally or nasally include incorporation of
the polypeptides into particles of biodegradable polymers (such as
acrylates or polyesters) by micro-encapsulation to aid uptake of
the microspheres from the gastrointestinal tract or nasal cavity
and to protect degradation of the proteins. Liposomes, ISCOMs,
hydrogels are examples of other potential methods which may be
further enhanced by the incorporation of targeting molecules such
as LTB, CTB or lectins (mannan, chitin, and chitosan) for delivery
of the antigenic polypeptides to the mucosal immune system. In
addition to the vaccine and the mucosal adjuvant or delivery system
the vaccine may include conventional pharmaceutically acceptable
carriers, excipients, fillers, coatings, dispersion media,
antibacterial and antifungal agents, buffers or diluents as
appropriate.
[0058] Another mode of this embodiment provides for either, a live
recombinant viral vaccine, recombinant bacterial vaccine,
recombinant attenuated bacterial vaccine, or an inactivated
recombinant viral vaccine which is used to protect against
infections caused by P. gingivalis. Vaccinia virus is the best
known example, in the art, of an infectious virus that is
engineered to express vaccine antigens derived from other
organisms. The recombinant live vaccinia virus, which is attenuated
or otherwise treated so that it does not caused disease by itself,
is used to immunise the host. Subsequent replication of the
recombinant virus within the host provides a continual stimulation
of the immune system with the vaccine antigens such as the
antigenic polypeptides, thereby providing long lasting
immunity.
[0059] Other live vaccine vectors include: adenovirus,
cytomegalovirus, and preferably the poxviruses such as
vaccinia.sup.(24) and attenuated salmonella strains.sup.(25-28).
Live vaccines are particularly advantageous because they
continually stimulate the immune system which can confer
substantially long-lasting immunity. When the immune response is
protective against subsequent P. gingivalis infection, the live
vaccine itself may be used in a protective vaccine against P.
gingivalis. In particular, the live vaccine can be based on a
bacterium that is a commensal inhabitant of the oral cavity. This
bacterium can be transformed with a vector carrying a recombinant
inactivated polypeptide and then used to colonise the oral cavity,
in particular the oral mucosa. Once colonised the oral mucosa, the
expression of the recombinant protein will stimulate the mucosal
associated lymphoid tissue to produce neutralising antibodies. For
example, using molecular biological techniques the genes encoding
the polypeptides may be inserted into the vaccinia virus genomic
DNA at a site which allows for expression of epitopes but does not
negatively affect the growth or replication of the vaccinia virus
vector. The resultant recombinant virus can be used as the
immunogen in a vaccine formulation. The same methods can be used to
construct an inactivated recombinant viral vaccine formulation
except the recombinant virus is inactivated, such as by chemical
means known in the art, prior to use as an immunogen and without
substantially affecting the immunogenicity of the expressed
immunogen.
[0060] As an alternative to active immunisation, immunisation may
be passive, i.e. immunisation comprising administration of purified
immunoglobulin containing an antibody against a polypeptide of the
present invention.
[0061] The antigenic polypeptides used in the methods and
compositions of the present invention may be combined with suitable
excipients, such as emulsifiers, surfactants, stabilisers, dyes,
penetration enhancers, anti-oxidants, water, salt solutions,
alcohols, polyethylene glycols, gelatine, lactose, magnesium
sterate and silicic acid. The antigenic polypeptides are preferably
formulated as a sterile aqueous solution. The vaccine compositions
of the present invention may be used to complement existing
treatments for periodontal disease.
[0062] A third aspect of the invention is a method of preventing or
treating a subject for periodontal disease comprising administering
to the subject a vaccine composition according to the present
invention.
[0063] In the present method a subject is treated including
prophylactic treatment for periodontal disease. Periodontal
diseases range from simple gum inflammation to serious disease that
results in major damage to the soft tissue and bone that support
the teeth. Periodontal disease includes gingivitis and
periodontitis. Bacteria, mainly Gram-negative species including P.
gingivalis cause inflammation of the gums that is called
"gingivitis." In gingivitis, the gums become red, swollen and can
bleed easily. When gingivitis is not treated, it can advance to
"periodontitis" (which means "inflammation around the tooth."). In
periodontitis, gums pull away from the teeth and form "pockets"
that are infected. The body's immune system tights the bacteria as
the plaque spreads and grows below the gum line. If not treated,
the bones, gums, and connective tissue that support the teeth are
destroyed. The teeth may eventually become loose and have to be
removed.
[0064] A fourth aspect of the invention is an antibody raised
against a polypeptide of the first aspect of the present invention.
Preferably, the antibody is specifically directed against the
polypeptides of the present invention.
[0065] In the present specification the term "antibody" is used in
the broadest sense and specifically covers monoclonal antibodies,
polyclonal antibodies, multispecific antibodies (e.g., bispecific
antibodies), chimeric antibodies, diabodies, triabodies and
antibody fragments. The antibodies of the present invention are
preferably able to specifically bind to an antigenic polypeptide as
hereinbefore described without cross-reacting with antigens of
other polypeptides.
[0066] The term "binds specifically to" as used herein, is intended
to refer to the binding of an antigen by an immunoglobulin variable
region of an antibody with a dissociation constant (Kd) of 1 .mu.M
or lower as measured by surface plasmon resonance analysis using,
for example a BIAcore.TM. surface plasmon resonance system and
BIAcore.TM. kinetic evaluation software (eg. version 2.1). The
affinity or dissociation constant (Kd) for a specific binding
interaction is preferably about 500 nM to about 50 pM, more
preferably about 500 nM or lower, more preferably about 300 nM or
lower and preferably at least about 300 nM to about 50 pM, about
200 nM to about 50 pM, and more preferably at least about 100 nM to
about 50 pM, about 75 nM to about 50 pM, about 10 nM to about 50
pM.
[0067] It has been shown that the antigen-binding function of an
antibody can be performed by fragments of a full length antibody.
Examples of binding fragments of an antibody include (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and
CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody; (v) a dAb fragment which consists of a
VH domain, or a VL domain; and (vi) an isolated complementarity
determining region (CDR). Furthermore, although the two domains of
the Fv fragment, VL and VH, are coded by separate genes, they can
be joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL,
and VH regions pair to form monovalent molecules (known as single
chain Fv (scFv). Other forms of single chain antibodies, such as
diabodies or triabodies are also encompassed. Diabodies are
bivalent, bispecific antibodies in which VH and VL domains are
expressed on a single polypeptide chain, but using a linker that is
too short to allow for pairing between the two domains on the same
chain, thereby forcing the domains to pair with complementary
domains of another chain and creating two antigen binding
sites.
[0068] Various procedures known in the art may also be used for the
production of the monoclonal and polyclonal antibodies as well as
various recombinant and synthetic antibodies which can bind to the
antigenic polypeptides of the present invention. In addition, those
skilled in the art would be familiar with various adjuvants that
can be used to increase the immunological response, depending on
the host species, and include, but are not limited to, Freud's
(complete and incomplete), mineral gels such as aluminum hydroxide,
surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, dinitrophenol, and potentially
useful human adjuvants such as Bacillus Calmette-Guerin (BCG) and
Corynebacterium parvum. Antibodies and antibody fragments may be
produced in large amounts by standard techniques (eg in either
tissue culture or serum free using a fermenter) and purified using
affinity columns such as protein A (e.g. for murine Mabs), Protein
G (eg for rat Mabs) or MEP HYPERCEL (eg for IgM and IgG Mabs).
[0069] Recombinant human or humanized versions of monoclonal
antibodies are a preferred embodiment for human therapeutic
applications. Humanized antibodies may be prepared according to
procedures in the literature.sup.(29,30), The recently described
"gene conversion metagenesis" strategy for the production of
humanized monoclonal antibody may also be employed in the
production of humanized antibodies.sup.(31). Alternatively,
techniques for generating the recombinant phase library of random
combinations of heavy and light regions may be used to prepare
recombinant antibodies.sup.(32).
[0070] The present invention also provides a composition useful in
the prevention or treatment of periodontal disease, the composition
comprising an antagonist of a P. gingivalis polypeptide of the
first aspect of the present invention and a pharmaceutically
acceptable carrier, wherein the antagonist inhibits P. gingivalis
infection.
[0071] As used herein, the term "antagonist" refers to a nucleic
acid, peptide, antibody, ligands or other chemical entity which
inhibits the biological activity of the polypeptide of interest. A
person skilled in the art would be familiar with techniques of
testing and selecting suitable antagonists of a specific protein,
such techniques would including binding assays. Possible
antagonists of PG0350, PG1374, PG1019 and PG0618 are preferably
antibodies, either monoclonal or polyclonal, which will inhibit the
binding of these proteins to host cells or other substrates or they
may be proteins or peptides that interfere with the binding of
these proteins.
[0072] The antibodies and antagonists of the present invention have
a number of applications, for example, they can be used as
antimicrobial preservatives, in oral care products (toothpastes and
mouth rinses) for the control of dental plaque and suppression of
pathogens associated with dental caries and periodontal diseases.
The antibodies and antagonists of the present invention may also be
used in pharmaceutical preparations (eg, topical and systemic anti
infective medicines).
[0073] In a sixth aspect of the present invention there is provided
a method of identifying a P. gingivalis polypeptide involved in the
progression of periodontal disease, wherein the method comprises
the steps of:
a) determining the relative amount of a polypeptide or peptide
thereof produced by P. gingivalis grown under haem limited
conditions; and b) determining the relative amount of the
polypeptide or peptide thereof produced by P. gingivalis grown
under higher haem conditions than step a); wherein an increase in
the amount of the polypeptide or peptide fragment thereof detected
in step a) compared to step b) indicates that the polypeptide is
involved in the progression of periodontal disease.
[0074] In order to grow P. gingivalis under haem limited
conditions, it is preferred that the concentration of haemin is
about 0.1 .mu.g/ml to about 0.5 .mu.g/ml. Haem limiting conditions
are achieved when the cell density of the P. gingivalis cells is
significantly lower than that observed under the growth conditions
of step (h) of the method of the invention. The higher haem
conditions of step (b) is preferably achieved using a concentration
of haemin of above 5 mg/ml.
[0075] A comparison of the relative amounts of a polypeptide in the
haem limited and higher haem conditions can be preferably
determined by using a differential proteomic approach. Preferably,
the amount of a polypeptide is determined by qualitative proteomic
analysis commonly used in the art, such as but not limited to, a
combined strategy of in-solution and in-gel digestion and LC-MS/MS,
analysis using stable isotope labelling strategies (ICAT) in
combination with MS.
[0076] The isolated antigenic P. gingivalis polypeptides identified
according to the method defined in the sixth aspect of the
invention can be used as targets for treating and preventing
periodontal disease. In particular, the isolated polypeptides can
be used to develop vaccine compositions against P. gingivalis, for
instance P. gingivalis infection, such as periodontal disease.
[0077] The present invention also provides interfering RNA
molecules which are targeted against the mRNA molecules encoding
the polypeptides of the first aspect of the present invention.
Accordingly, in a seventh aspect of the present invention there is
provided an interfering RNA molecule, the molecule comprising a
double stranded region of at least 19 base pairs in each strand
wherein one of the strands of the double stranded region is
complementary to a region of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID
NO: 7 or SEQ ID NO: 8.
[0078] So called RNA interference or RNAi is well known and further
information regarding RNAi is provided in Hannon (2002) Nature 418:
244-251, and McManus & Sharp (2002) Nature Reviews; Genetics
3(10): 737-747, the disclosures of which are incorporated herein by
reference.
[0079] The present invention also contemplates chemical
modification(s) of siRNAs that enhance siRNA stability and support
their use in vivo (see for example, Shen et al. (2006) Gene Therapy
13: 225-234). These modifications might include inverted abasic
moieties at the 5' and 3' end of the sense strand oligonucleotide,
and a single phosphorthioate linkage between the last two
nucleotides at the 3' end of the antisense strand.
[0080] It is preferred that the double stranded region of the
interfering RNA comprises at least 20, preferably at least 25, and
most preferably at least 30 base pairs in each strand of the double
stranded region. The present invention also provides a method of
treating a subject for periodontal disease comprising administering
to the subject at least one of the interfering RNA Molecules of the
invention.
[0081] In order that the nature of the present invention may be
more clearly understood preferred forms thereof will now be
described with reference to the following example.
Example 1
1. Materials and Methods
1.1 Bacterial Strain and Chemicals
[0082] P. gingivalis W50 (ATCC 53978) was obtained from the culture
collection of the Centre for Oral Health Science, The University of
Melbourne. Chemicals used were ultra high purity except for MS work
where LC MS grade reagents were used (Sigma, Reidel-de Ha{umlaut
over (c)}n).
1.2 Growth and Harvesting of P. gingivalis
[0083] P. gingivalis W50 was grown in continuous culture using a
Bioflo 110 fermenter/bioreactor (New Brunswick Scientific) with a
400 mL working volume. The growth medium was 37 g/mL brain heart
infusion medium (Oxoid) supplemented with 5 mg/mL filler sterilized
cysteine hydrochloride, 5.0 .mu.g/mL haemin (haem-excess) or 0.1
.mu.g/mL haemin (haem-limited). Growth was initiated by inoculating
the culture vessel with a 24 h batch culture (100 mL) of P.
gingivalis grown in the same medium (haem-excess). After 24 h of
batch culture growth, the medium reservoir pump was turned on and
the medium flow adjusted to give a dilution rate of 0.1 h.sup.-1
(mean generation time (MGT) of 6.9 h). The temperature of the
vessel was maintained at 37.degree. C. and the pH at 7.4.+-.0.1.
The culture was continuously gassed with 5% CO.sub.2 in 95%
N.sub.2. Cells were harvested during steady state growth, washed
three times with wash buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5
mM MgCl.sub.2) at 5000 g for 30 min and disrupted with 3 passes
through a French Pressure Cell (SLM, AMINCO) at 138 MPa. The lysed
cells were then centrifuged at 2000 g for 30 min to remove unbroken
cells followed by ultracentrifugation at 100000 g, producing a
soluble (supernatant) and membrane fraction. All fractions were
carried out on ice.
1.3 Preparation of Samples for Non-Quantitative Proteome
Analysis
[0084] Non-quantitative proteome analysis was carried out using two
methods, in-solution digestion with trypsin followed by LCMS with
gas phase fractionation (GPF) and in-gel digestion followed by LCMS
(geLC-MS) as part of a combined strategy (FIG. 1). For the
in-solution digestion method, protein was boiled at 95.degree. C.
for 3 min, precipitated with TCA (16%) and resuspended in
solubilization buffer (8 M Urea, 50 mM Tris-HCl pH 8.3, 5 mM EDTA,
0.05% SDS). Protein concentration was determined with a BCA,
protein reagent (Pierce) and adjusted to 2 .mu.g/.mu.L. Reduction
was carried out with 1 in M DTT for 30 min and alkylation using 10
mM iodoacetamide for 60 min. The solution was diluted with water to
a final concentration of 1 M urea before digestion. Digestion was
carried out using sequencing-grade modified trypsin (Promega) at a
ratio of 1:100 w/w trypsin to protein at 37.degree. C. for 16 h.
The digestion was terminated by formic acid addition to a final
concentration of 1% v/v. Peptides were then desalted using Sep-Pak
C18 cartridges (Waters), dried using a vacuum centrifuge
(Thermosavant) and resuspended in 5% acetonitrile in 0.1% TFA. An
amount of peptide equivalent to 2 .mu.g was injected for each
LC-MS/MS analysis.
[0085] For the geLC-MS method, 25 .mu.g of protein was separated on
a precast Novex 12% Tris-HCl glycine gel (Invitrogen) and stained
overnight with Commassie Brilliant Blue G-250 (Sigma). The gel was
divided into thirty individual sections which were then excised and
cut into approximately 1 mm.sup.3 cubes. Destaining was carried out
three times with a solution of 50% ethanol and 25 mM ammonium
bicarbonate (ABC) buffer followed by dehydration with 100% ethanol.
Reduction was carried out by incubating the dehydrated gel cubes
with 10 nM DTT in 25 mM ABC for 60 min at 56.degree. C. The
reduction solution was then replaced with 55 mM of iodoacetamide in
25 mM ABC and incubated for 45 min. The gel cubes were washed twice
in 50 mM ABC and dehydrated with 100% ethanol. Thirty .mu.L of
modified sequencing-grade trypsin at a concentration of 5 .mu.g/mL
in 25 mM ABC buffer and 1 mM CaCl.sub.2 was added and incubated at
4.degree. C. for 30 min. Excess trypsin solution was removed and 15
.mu.L of 25 mM ABC buffer was added. Digestion was carried out
overnight at 37.degree. C. and terminated by TFA addition to a
final concentration of 0.1% v/v. The supernatant was then
transferred to an eppendorf tube. To the gel pieces 50 .mu.L, of
50% ethanol in 0.1% TFA was added and sonicated for 15 min. The
process was repeated and all supernatants derived from one gel
section were pooled and dried to about 10 .mu.L using a vacuum
centrifuge.
1.4 Preparation of Samples for Quantitative ICAT Analysis
[0086] Protein labelling and separation were based on the
geLC-MS/MS approach.sup.(33) using the cleavable ICAT reagent
(Applied Biosystems). Protein was first precipitated using TCA
(16%) and solubilised with 6 M urea, 5 mM EDTA, 0.05% SDS and 50 mM
Tris-HCl pH 8.3. Protein concentration was determined using the BCA
protein reagent and adjusted to 1 mg/ml. 100 .mu.g of protein from
each growth condition was individually reduced using 2 .mu.L of 50
mM Tris(2-carboxy-ethyl)phosphine hydrochloride for 1 h at
37.degree. C. Reduced protein from the haem-limitation growth
condition was then alkylated with the ICAT.sub.heavy reagent and
protein from haem-excess growth condition with the ICAT.sub.light
reagent. The two samples were then combined and subjected to
SDS-PAGE on a precast Novex 10% NUPAGE gel (Invitrogen). The gel
was stained for 5 min using SimplyBlue.TM. SafeStain (Invitrogen)
followed by destaining with water. The gel lane was then excised
into 20 sections from the top of the gel to the dye front.
[0087] The excised sections were further diced into 1 mm.sup.3
cubes and in-gel digested overnight and extracted twice according
to the above procedure. The pooled supernatant was dried under
reduced vacuum to about 50 .mu.L followed by mixing with 500 .mu.L,
of affinity load buffer before loading onto the affinity column as
per manufacturer's instruction (Applied Biosystems). Eluted
peptides were dried and the biotin tag cleaved with neat TFA at
37.degree. C. for 2 h. followed by drying under reduced vacuum. The
dried samples were suspended in 35 .mu.L of 5% acetonitrile in 0.1%
TFA.
1.5 Liquid Chromatography and Mass Spectrometry
[0088] MS was carried Out using an Esquire HCT ion trap mass
spectrometer (Bruker Daltonics) coupled to an UltiMate Nano LC
system (LC Packings--Dionex). Separation was achieved using a LC
Packings reversed phase column (C18 PepMap100, 75 .mu.m
i.d..times.15 cm, 3 .mu.m, 100 .ANG.), and chilled in 0.1% formic
acid with the following acetonitrile gradient: 0-5 min (0%), 5-10
min (0-10%), 10-100 min (10-50%), 100-120 min (50-80%), 120-130 min
(80-100%).
[0089] The LC output was directly interfaced to the nanospray ion
source. MS acquisitions were performed under an ion charge control
of 100000 in the m/z range of 300-1500 with maximum accumulation
time of 100 ms. When using GPF three additional m/z ranges
(300-800, 700-1200 and 1100-1500) were used to select for precursor
ions and each m/z range was carried out in duplicate to increase
the number of peptides identified. MS/MS acquisition was obtained
over a mass range from 100-3000 m/z and was performed on up to 10
precursors for initial complete proteome analysis and 3 for ICAT
analysis for the most intense multiply charged ions with an active
exclusion time of 2 rain.
1.6 Protein Identification for Non-Quantitative Proteome
Analysis
[0090] Peak lists were generated using DataAnalysis 3.2 (Bruker
Daltonics) using the Apex peak finder algorithm with a compound
detection threshold of 10000 and signal to noise threshold of 5. A
global charge limitation of +2 and +3 were set for exported data.
Protein identification was achieved using the MASCOT search engine
(MASCOT 2.1.02, Matrix Science) on MS/MS data queried against the
P. gingivalis database obtained from The Institute for Genomic
Research (TIGR) website (www.tigr.org). The matched peptides were
further evaluated using the following criteria, i) peptides with a
probability based Mowse score corresponding to a p-value of at most
0.05 were regarded as positively identified, where the score is
-log.times.10(log(P)) and P is the probability that the observed
match is a random event ii) where only one peptide was used in the
identification of a specific protein and the MASCOT score was below
30, manual verification of the spectra was performed.
1.7 Protein Identification for ICAT
[0091] To increase confidence in the identification of ICAT-labeled
proteins especially for those with single peptide hits, additional
filters were applied as follows: i) the heavy and light peptides of
an ICAT pair must have exhibited closely eluting peaks as
determined from their extracted ion chromatograms ii) for proteins
with a single unique peptide, this peptide must have been
identified more than once (e.g in different SDS-PAGE fractions or
in both the light and heavy ICAT forms iii) if a single peptide did
not meet the criteria of (ii), the MASCOT score must have been
.gtoreq.25, the expectation value .ltoreq.0.01 and the MS/MS
spectrum must have exhibited a contiguous series of `b` or `y`-type
ions with the intense ions being accounted.
1.8 Estimation of False Positive
[0092] To independently estimate the level of false positive
assignments, a reverse database of P. gingivalis was created by
reversing the order of the amino acid sequences for each protein
such that the database is identical in size to the normal database
in terms of the protein number, size and distribution of amino
acids.sup.(34). The false positive rate was thus estimated as
N.sub.R/N.sub.F where N.sub.R=number of peptides identified with
the reverse database (MASCOT score of peptide above threshold for
the reverse database) and N.sub.F=number of peptides identified
with the normal database (MASCOT score of peptide above threshold
for normal database). False positives were determined from the
comprehensive proteome analysis (N.sub.F=18375 peptides) and
quantitative ICAT analysis (N.sub.F=530 peptides).
1.9 Quantification of Relative Abundance
[0093] The ratio of isotopically heavy .sup.13C to light .sup.12C
ICAT labelled peptides was determined using a script from
DataAnalysis (Bruker Daltonics) and verified manually based on
measurement of the monoisotopic peak intensity (signal intensity
and peak area) in a single MS spectrum. The minimum ion count of
parent ions used for quantification was 2000 although >96% of
both heavy and light precursor ions were >10000. In the case of
poorly resolved spectra, the ratio was determined from the area of
the reconstructed extracted ion chromatograms (EIC) of the parent
ions. Averages were calculated for multiple peptides derived from a
single parent protein and outliers were removed using the Grubb's
test with .alpha.=0.05.
1.10 Genome Analysis
[0094] The cellular localisation of P. gingivalis proteins was
predicted using CELLO (http://cello.life.nctu.edu.tw.sup.(35) and
transmembrane helices using TMHMM 2.0
(www.cbs.dtu.dk/services/TMHMM-2.0) based on the sequence obtained
from TIGR. To estimate the relative expression level of the
proteins identified as compared to the theoretical proteome CAI
values were calculated based on the coding sequence of P.
gingivalis from genebank (ftp
://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/Porphyromonas_gingivalis_W83-
/) using the program INteractive Codon Analysis 1.12a
(http://www.bioinfo-hr.org/inca,.sup.(14) with ribosomal proteins
and tRNA synthases being defined as highly expressed genes. Operon
prediction was carried out from the Microbesonline website
(http://microbesonline.org.sup.(36).
1.11 Construction of ECR312 Mutant
[0095] P. gingivalis W50 Open Reading Frame PG1374 potentially
encodes an immunoreactive 47 KDa antigen (PG97) based on the P.
gingivalis W83 genome (www.tigr.org). To construct P. gingivalis
PG1374 mutant, a 672 bp upstream fragment of the PG1374 gene with
flanking ApaI and AatII restriction sites (underlined) was
generated from the W50 genomic DNA by PCR with primers
ECR312ApaI-For (5'-AGAGGGCCCTAGCAATCATTGCATTGCT-3') and
ECR312AatII-Rev (5'-TGCGACGTCGTGTTACCAATAGAGGATT-3'). This fragment
was cloned into AatII and BamHI sites on pAL30, pGem.RTM.T-easy
(Promega) containing a subcloned ermF cassette.sup.(37) to create
pAL36. Similarly, a 565 bp fragment downstream of PG1374 with
flanking PstI and NdeI restrictions sites was amplified with
ECR312PstI-For2 (5'-TGACTGCAGGCTTTCGACCTTGGATCTT-3') and
ECR312NdeI-Rev2 (5'-TCGCATATGAAGAAATAAGTGCCGTCGG-3') primers, and
cloned into PstI and NdeI restrictions sites in pAL36. The
resulting plasmid having ermF cassette flanked with upstream and
downstream fragments of the PG1374 open reading frame (designated
as pAL36.1) was linearized with Scar and transformed into P.
gingivalis W50 as previously described.sup.(38). Transformed cells
were selected after 7 days of incubation at 37.degree. C. under
anaerobic conditions on HBA plate containing 10 .mu.g mL.sup.-1
erythromycin. Confirmation of DNA integration was performed by PCR
analysis and the resulting mutant was designated as ECR312.
1.12 Antibiotic Protection Invasion Assay
[0096] To compare the invasion efficiencies of W50 and ECR312, an
antibiotic protection assay was carried out as described previously
in a 24-well cell culture plate.sup.(39). Briefly, a fixed number
of P. gingivalis cells were allowed to invade a KB monolayer
(.about.10.sup.5 cells in each well). Non invaded or adhered cells
were killed by further incubation for 1 h with gentamicin (300
.mu.g/mL) and metronidazole (200 .mu.g/mL). Colony forming units of
invaded bacteria were then enumerated on horse blood agar
plates.
1.13 Cell Binding Assays
[0097] The cell binding assay was carried out as described
previously.sup.(40). Briefly, P. gingivalis was first grown to mid
log phase to a cell density of .about.2.9.times.10.sup.9 cells/mL.
The cells were then washed followed by labelling with 500 .mu.g of
fluorescein isothiocyanate (FITC) (Invitrogen) resuspended in 500
.mu.L DMSO followed by incubation at 37.degree. C. for 45 min with
shaking. After incubation, the cells were further washed,
resuspended in incomplete Earl's minimum essential medium (JRH
Biosciences) and the P. gingivalis cells adjusted based on cell
counts using a FACSCaliber flow cytometer (Becton Dickinson, San
Jose, Calif.). The green emission of FITC was measured with a
525-nm filter (FL1). The multiparametric data were analyzed using
CellQuest software (Becton Dickinson, San Jose, Calif.). All
measurements were done in duplicate, and for quantitation of FITC
fluorescence, mean fluorescence intensity (MFI) values were
used.
[0098] Binding of the wild type P. gingivalis and ECR312 was
carried out in parallel by inoculating 200 .mu.L of cell suspension
onto the KB cells at 5% CO.sub.2 atmosphere at 37.degree. C. for 40
min. Following incubation the supernatant containing the KB cells
and bacteria were transferred to a 1.5 mL tube. The remaining bound
cells were then detached off the well with 200 .mu.L of
Trypsin-EDTA mixture (JRH Bioscience) for 5 min at 37.degree. C.
and pooled with the corresponding collected supernatants. 500 .mu.L
of complete EMEM was then added to inactivate the trypsin followed
by three washes and final suspension in 1 mL PBS. The bound cells
were counted on the flow cytometer as described earlier.
2. Results and Discussion
[0099] 2.1 Growth of P. gingivalis in Continuous Culture
[0100] When grown in continuous culture in a rich medium containing
excess haem P. gingivalis W50 achieved a steady state cell density
approximately 48 h after inoculation of 2.03.+-.0.04 mg cellular
dry weight/mL. When the concentration of haemin in the growth
medium was decreased from 5.0 .mu.g/ml to 0.1 .mu.g/ml, a
significantly lower steady state cell density of 0.99.+-.0.20 mg
cellular dry weight/mL was achieved demonstrating that haem
availability was limiting growth. The effect of haem-limitation on
cell density was alleviated when haem was added back into the
culture.
[0101] The growth of P. gingivalis during haem-limitation was in
agreement with previous studies using chemostats with a P.
gingivalis mean generation time of 6.9 h.sup.(41,42). Due to the
inability of P. gingivalis to synthesize PPIX, this essential
nutrient is thought to be acquired through proteolysis of
haemoglobin and other haem containing plasma proteins.sup.(9) and a
deficiency in haem was thus reflected in the significantly lower
cell density.
2.2 Proteome Analysis of P. gingivalis Grown Under
Haem-Limitation
[0102] The proteome of P. gingivalis grown under haem-limitation
was extensively analysed by two different approaches. Using
in-solution digestion followed by LCMS with gas phase
fractionation, 344 proteins were identified. In the geLCMS
approach, 385 proteins were identified while 247 proteins were
found by both approaches. With the combined strategy a total of 478
proteins were identified (see Table 1) with an estimated false
positive rate of 0.4% calculated from searches against the P.
gingivalis reverse database. 77.0% of all proteins were identified
by .gtoreq.2 unique peptides or by .gtoreq.2 identical peptides
from independent LCMS runs (from different m/z ranges or SDS PAGE
bands). The 478 identified proteins represent .about.25% of all the
1988 assigned protein-encoding genes identified by whole-genome
analysis.sup.(43). Although a quarter of the total predicted
proteome was identified this figure is higher if the actual number
of genes expressed during any one growth condition is taken into
account. In Pseudomonas aeruginosa and Bacillus subtilis the
percentage of total ORFs transcribed during a single growth
condition was estimated to be 60% and 40%,
respectively.sup.(44,45). By examining the duty cycle limitation of
their mass spectrometers Zhang and co-workers.sup.(46) estimated
that around 60% of P. gingivalis predicted ORFs were expressed
under their growth condition. Therefore based on these figures
between 41-62% of P. gingivalis proteins expressed under the
present growth conditions have been identified. The functional
classification of the 478 identified proteins is shown in Table
1.
TABLE-US-00002 TABLE 1 Coverage of the theoretical proteome of P.
gingivalis using the combined strategy shown in FIG. 1. Proteins
ID/ % of Functional class of proteins.sup.a Total Protein.sup.b
class ID Energy metabolism 78/140 55.7 Protein synthesis 68/117
58.1 Fatty acid and phospholipid metabolism 9/16 56.3 Protein fate
37/75 49.3 Purines, pyrimidines, nucleosides, and 22/44 50.0
nucleotides Central intermediary metabolism 11/23 47.8 Cellular
processes 12/46 26.1 Cell envelope 32/140 22.8 Amino acid
biosynthesis 2/19 10.5 Signal transduction 2/12 16.6 Unknown
function 47/201 23.4 Transcription 7/31 22.6 Transport and binding
proteins 20/119 16.8 DNA metabolism 14/81 17.3 Biosynthesis of
cofactors, prosthetic groups, 14/88 15.9 and carriers Hypothetical
proteins (includes conserved) 96/695 13.8 Regulatory functions 5/47
10.6 Other categories 2/134 1.5 .sup.aFunctional classification
data obtained from TIGR (www.tigr.org) .sup.bSome proteins have
been assigned to more than one functional class
[0103] To date, there is only one reported attempt to identify P.
gingivalis proteins globally using a multidimensional proteomics
approach.sup.(46). The present inventors have identified 478 P.
gingivalis W50 proteins that were expressed during continuous
culture under haem-limitation compared to the study of Zhang and
co-workers.sup.(46) where 1014 P. gingivalis ATCC 33277 proteins
were identified when this strain was cultured in keratinocyte
growth medium and the same medium exposed to secreted epithelial
cell components. As the strain used, the growth conditions and the
processing of MS data were different in the two studies, it is
difficult to make direct comparisons between the two datasets.
Nevertheless, 75 proteins were uniquely identified in the present
methods.
[0104] Using CELLO to predict the subcellular protein localisation,
most of the proteins identified in this study were predicted to be
from the cytoplasm (347 out of a predicted 1350 proteins), followed
by the periplasmic space (48/113), outer membrane (47/154), inner
membrane (24/256) and extracellular (12/35). As expected, a low
percentage of predicted inner membrane proteins were identified. To
further increase the confidence of predicting inner membrane
proteins the Transmembrane Hidden Markov Model (TMHMM) was used.
Using the TMHMM approach 20 out of the 242 proteins predicted to
have >2 transmembrane domains (TMD) were identified and 5 out of
44 proteins predicted to have >10 TMDs were identified. Notably,
all 12 of the membrane proteins with >10 transmembrane domains
detected were identified from the in-solution digestion method.
2.3 Significance of Identified Proteins
[0105] Highly expressed genes in many bacteria often have a strong
composition bias in terms of codon usage. The Codon Adaptation
Index (CAD can be used to predict the expression level of a gene
based on its codon sequence, with a higher CAI value indicating
increased expression.sup.(47). Of 1685 genes in the P. gingivalis
genome that have >100 codons, almost 92% have CAI values between
0.62 and 0.80 (FIG. 2). Although this range is narrow compared with
eukaryotic organisms.sup.(48) it is similar to the thermophile
Thermoanaerobacter tengcongensis where 89% of the predicted genes
have CAI values between 0.35 and 0.50.sup.(49). The CAI values of
the genes encoding the 478 identified proteins in this study have a
similar distribution to the theoretical proteome although there was
a bias towards the detection of higher abundance proteins. Despite
this bias, a number of proteins encoded by genes of very low CAI
were identified. This result clearly exemplifies the problem of the
large dynamic range of protein abundance in cells, showing it is
currently not possible to detect all proteins at once.
[0106] The functional classes of proteins with the highest
percentage of identified proteins are those involved in energy
metabolism (Table 1), typically those involved in fermentation
(95%, CAI 0.70-0.80), glycolysis (82%, CAI 0.71-0.83) and
metabolism of amino acids and amines (81%, CAI 0.71-0.84). This was
largely expected as essential proteins involved in basic metabolic
functions such as energy metabolism have been shown to be very
abundant in the bacterial cell. Most importantly almost 90% of
these proteins are predicted to be in the cytoplasm, which also
made detection easier compared with membrane proteins. The
functional classes of proteins represented least are those involved
in transpositioning, hypothetical proteins and regulatory
functions.
[0107] Although the complete genome of P. gingivalis has been
sequenced, many critical questions regarding cellular functions
remain unanswered. Proteomic studies that identify the translated
gene products therefore help provide additional insights into the
functional genome. For example P. gingivalis is known to be
asaccharolytic.sup.(3) although the genome contains putative ORFs
for all enzymes of the glycolytic pathway.sup.(43). The poor
utilization of this pathway has been attributed to the glucose
kinase gene being interrupted by an insertion element.sup.(43). In
keeping with this finding glucose kinase, or another
glycolysis-specific enzyme, phosphofructokinase was not identified.
In contrast, all enzymes involved in gluconeogenesis were found,
suggesting glucose necessary for processes such as polysaccharide
biosynthesis may be derived via this pathway.
2.4 Response of P. gingivalis to Haem-Limitation as Determined
Using ICAT
[0108] To carry out the quantitative ICAT analysis of the P.
gingivalis response to haem-limitation, the geLCMS approach was
chosen, as the in-solution ICAT method was unsatisfactory due to
the presence of strong interfering triply charged ions. The
presence of these triply charged ions resulted in very low number
of protein identifications. The ICAT labelled soluble and insoluble
protein fractions were therefore independently separated by
SDS-PACE and each gel lane divided into 20 sections for in-get
tryptic digestion followed by affinity purification and LCMS. In
total 142 proteins were identified. No matches to the reverse
database were obtained indicating a low level of false positive
identification. Considering proteins detected in both fractions, 53
proteins (34.0%) were identified based on the presence of two or
more unique peptides with a probability based Mowse score
corresponding to a p-value of at most 0.05, 60 proteins (38.5%)
were identified based on the presence of one unique peptide
identified from two or more different fractions or both ICAT
labelling states (of those 58 have MASCOT score of .gtoreq.25) and
43 of the proteins (27.5%) were identified on the basis of a single
unique peptide having a MASCOT score .gtoreq.25, expectation value
of .ltoreq.0.01, a contiguous series of `b` or `y`-type ions and
the intense ions being accounted for, when interpreted manually. An
example of a protein identification based on the analysis of a
single unique peptide is shown in FIG. 3.
[0109] Of the proteins identified, 103 were found in the soluble
fraction, 53 in the insoluble fraction and 14 proteins in both
fractions. In response to the change in environmental conditions
from haem-excess to haem-limitation 70 of the identified proteins
exhibited at least a two-fold change in abundance (FIG. 4). Of
these, the abundance of 53 proteins increased more than 2 fold and
the abundance of 17 proteins decreased more than 2 fold during
haem-limitation.
[0110] In order to assess the reproducibility of the present data
and to increase protein identification for the insoluble fractions,
cells were harvested from the chemostat on different days during
haem-limitation and excess growth. A different extraction method
followed by ICAT labelling was then repeated with modified
protocols (not shown). The data obtained were reproducible with
proteins showing similar abundance ratios for selected proteins
shown in Table 2 (e.g. PG0695 L/H=1.1, relabelling=1.3; PG0350
L/H=3.2, relabelling=2.6; PG0159 L/H=2.0, relabelling=2.2; PG0232
L/H=0.4, relabelling=0.4). To further verify the data, the relative
abundance of those identified proteins that are predicted to be
encoded by genes forming an operon were compared.sup.(50). Five
groups of proteins were found to be encoded by predicted operons or
by genes grouped at specific loci (Table 2--shaded). In each case
the abundance of the encoded proteins appeared to be similar. One
of the predicted operons encodes the outer membrane proteins, Omp40
(PG0694) and Omp41 (PG0695) whose abundance were unchanged at a
ratio of 1.1 (haem-limitation/haem-excess, L/E). These proteins
have high sequence similarity to the OmpA-like porins of
Gram-negative bacteria.sup.(51) and are thought to provide a
physical linkage between the outer membrane and the peptidoglycan
layer. These structural proteins would not be expected to vary in
abundance with a change in environmental haem levels. The remaining
four predicted operons were found to be associated with glutamate
or aspartate catabolism.
TABLE-US-00003 TABLE 2 Expression data of selected proteins in P.
gingivalis during growth in haem-limitation Tigr Protein and
peptide n- Fold SD No Acc# sequence identified Score.sup.1 N.sup.2
ICAT.sup.3 change.sup.4 (.+-.) Proteinases 1 PG2024/
Arginine-specific protease 11 2 0.39 0.1 PG0506 (RgpA.sub.Cat/RgpB)
.GQDEMNEILC*EK 51/14 .C*YDPGVTPK 24/14 2 PG2024/ Arginine-specific
protease 19 3 0.95 0.2 PG0506 (RgpA/RgpB adhesins)
.DAGMSAQSHEYC*VEVK 34/15 .EGLTATTFEEDGVAAG 44/13 NHEYC*VEVK
.C*VNVTVNSTQFNPVK 59/15 3 PG0232 Zinc carboxypeptidase 4 1 0.40 0.1
.C*QILIENHDKR 21/18 .YPSLC*TTSVIGK 56/19 4 PG0026 Hypothetical
protein 5 2 0.46 0.1 (Homology to Arg proteases) .C*VVNSPGGQTASMAK
30/14 .FSNLPVLGGESC*R 58/14 Invasion related proteins 5 PG0350
Internalin related protein 11 4 3.2 0.6 .FVPYNDDEGGEEENVC 35/13
*TTEHVEMAK .ILMELSEADVEC*TIK 46/14 .ILHC*NNNQLTALNLSA 23/15 NTK
.LDLPANADIETLNC*SK 52/13 6 PG1374 Immunoreactive 47KDa 5 2 6.5 0.7
protein .GLSVLVC*IISNQIAGEE 27/15 MTK .NPNLTYLAC*PK 61/13 7 PG0159
Endopeptidase PepO 6 1 2.0 0.3 .METELAQIC*YSK 55/13 8 PG2132
Fimbrillin FimA 2 1 0.50 -- .YDASNELRPTILC*IYGK 45/16 Iron
transport and related proteins 9 PG1552 HmuR 1 1 4.0 --
.MNSDELFEEITYPGYTIC*R 25/15 10 PG1019 Hypothetical protein 2 1 25.0
-- .TYMIDTNDSENDC*IAR 70/14 11 PG1286 Ferritin 2 1 1.2 --
.FGSVLEVFQQVYEHEC*K 73/13 12 PG0090 Dps family protein 3 1 1.1 0.1
.EEHELVC*AASTLK 36/13 13 PG0618 Alkyl hydroperoxide 1 1 41.6 --
reductase subunit C 36/15 .AAQYVAAHDGQVC*PAK Others 14 PG0694 Omp40
5 1 1.1 0.1 .RPVSC*PECPEPTQPTVTR 26/16 15 PG0695 Omp41 12 1 1.1 0.1
.RPVSC*PECPEVTPVTK 39/15 .sup.1Highest scoring peptide
score/threshold score (P = 0.05) .sup.2Total number of independent
peptide identification events for each protein .sup.3Numbcr of
unique ICAT labelled peptides identified for each protein
.sup.4Average ratios of all quantified peptides for each protein in
fold change (Haem-limitation/excess) *Denotes ICAT modified
cysteine
2.5 Host Cell Invasion Related Proteins
[0111] Three proteins possibly involved in invasion of host cells,
internalin related protein (PG0350), immunoreactive 47 kDa protein
(PG1374) and endopeptidase PepO (PG0159) were higher in abundance
during haem-limitation (Table 2). During an antibiotic protection
invasion assay P. gingivalis lacking a functional PG1374 had
approximately 50% lower invasion capability into epithelial cells
as compared to the wild type (W50, 32625.+-.2582 cfu/mL, ECR312
16250.+-.1089 cfu/mL; p<0.01, Student's T-test). In a separate
binding assay, there is no significance difference in the adherence
of the PG1374 mutant as compared to the wild type W50 (FIG. 5).
[0112] PG1374 and PG0350 belong to a new class of cysteine
containing protein with leucine rich repeat domains similar to the
L. monocytogenes internalin protein. In1J.sup.(52). In L.
monocytogenes, there are at least fifteen members of the internalin
family and all have been found to share certain structural features
consisting of a signal peptide, N-terminal leucine rich repeat
domain followed by a conserved inter-repeat region. Many of these
proteins are involved in the cellular invasion process.sup.(53). It
has not been demonstrated why multiple internalins exist, but they
are proposed to confer tropism toward different cell
types.sup.(54).
[0113] The higher abundance of PG1374 and PG0350 during haem
limitation (6.5 and 3.2 fold increase respectively) in the current
work suggests the expression of these two proteins is stimulated
during low haem growth conditions. From the sequence information
and predicted structure, more than half of the internalin LRR
residues face outwards and are variable, suggesting them to be for
protein-protein interaction surfaces specific to the different
internalin classes.sup.(55). PG1374 and PG0350 both possesses a
signal peptide and are part of the novel class of up to 34 cell
surface-located outer membrane proteins that have no significant
sequence similarities apart from a conserved C-Terminal Domain
(CTD) of approximately 80 residues.sup.(56). In addition PG1374 is
strongly immunogenic when probed with sera from human periodontitis
patients.sup.(57) which further suggests it to be involved in cell
surface protein interactions.
[0114] The process of internalization of P. gingivalis into
gingival epithelial cells is thought to involve a coordinated
process of attachment and invasion mediated by fimbriae and a
variety of cell surface proteinases.sup.(58-60). A P. gingivalis
33277 mutant lacking a functional putative internalin (PG0350) was
shown to exhibit similar invasive characteristics but reduced
biofilm formation capability compared to the wild type
bacteria.sup.(46,61). The similar invasion was attributed to the
presence of fimbriae that also play a role in epithelial cell
invasion by strain 33277 although there is also a possibility that
the similar invasiveness of this mutant was due to the presence of
a second putative internalin protein (PG1374) encoded in the P.
gingivalis genome. A double knockout of these two putative
internalin proteins would potentially shed light on their possible
cooperative invasive roles.
[0115] The 50% reduction in epithelial cell invasion by ECR 312 and
no difference in cell binding clearly demonstrate that the observed
reduced invasion into epithelial cells by P. gingivalis deficient
in PG1374 is not due to lesser adherence but a real defect in the
invasion process (FIG. 5). Bacterial invasion has been shown to be
a highly complex process involving numerous proteins and
receptors.sup.(62). The involvement of multiple factors involved in
P. gingivalis invasion has been demonstrated by Lamont's group at
the University of Florida and includes haloaeid dehalogenase,
endopeptidases, a cation-transporting ATPase and an ATP-binding
cassette transporter.sup.(60,63). The discovery of PG1374 as an
epithelial cell invasion related protein therefore adds to the list
of proteins involved in this complex host cell invasion process by
bacterial pathogens.
[0116] For many bacterial pathogens, it has been well established
that iron availability influences virulence and the invasion
process, but little is known about the influence of haem on the
expression of P. gingivalis invasion genes. We were unable to
perform binding and invasion assay on haem-limited cells because of
the high mortality rate of the P. gingivalis from the oxidative
stress likely due to lack of the protective layer of the .mu.-oxo
bis-haem form of iron PPIX.sup.(64-66). However in this study, we
have shown that the levels of the putative invasion related
proteins PepO, PG0350 and PG1374 increased under haem-limitation
and PG1374 is involved in the cell invasion.
[0117] These experiments described above are the first quantitative
proteomic analysis of the response of P. gingivalis to a change in
environmental conditions and demonstrates the utility of the stable
isotope labelling approach combined with complete proteome
analyses. P. gingivalis responds to limitation of the essential
micronutrient haem by increasing the abundance of a number of
proteins linked to the oxidative stress response, virulence and
invasion of host cells.
2.6 Cell Surface Located Protein PG1019
[0118] A P. gingivalis hypothetical protein, PG1019 was observed to
be 25 times more abundant when the bacterium was grown under
haem-limitation. Bioinformatic analyses suggest that PG1019 is a
lipoprotein that is encoded by a gene located immediately upstream
of a gene encoding a putative outer membrane receptor protein
(PG1020) in a predicted operon. Multiple alignment (not shown) of
PG1020 with known P. gingivalis TonB-linked outer membrane
receptors shows the presence of a putative TonB box (residues
118-126), that is one of the characteristics of TonB-linked
receptors.sup.(67) and a conserved region (residues 236-272) which
Simpson and co-workers.sup.(68) refer to as the TonB box IV region.
TonB-linked outer membrane receptors have been implicated with many
iron, iron complex and other micronutrient uptake systems. The high
abundance of PG1019 under haem limited growth conditions would be
consistent with this protein being an accessory lipoprotein to a
Ton-B linked system involved in the transport of iron/iron
complexes into the cell or the sensing of environmental iron or
iron complexes, although this remains to be demonstrated. In
addition to the proteomic data a transcriptomic analysis using
custom made P. gingivalis DNA microarrays of P. gingivalis W50
compared to a mutant lacking a function feoB1 gene (P. gingivalis
FB1) was performed. The wild type W50 and FB1 mutant were both
grown in continuous culture in haem excess conditions. P.
gingivalis FB1 has approximately half the cellular iron content of
the wild type W50.sup.(69). Genes that show an increase in
transcription are therefore likely to be upregulated in response to
the decrease in intracellular or environmental iron content. Both
PG1019 and PG1020 were significantly upregulated to similar levels
in the FB1 mutant compared to the wild type. PG1019 showed a Log 2
increase of 2.46 and PG1020 showed a Log 2 increase of 2.33 at a
significance level of P<0.05 in biological replicates, this is
further evidence that these genes are located in an operon. Further
a separate transcriptomic DNA microarray analysis of P. gingivalis
W50 indicated that there was little or no expression of the PG1019
gene during haem-excess growth.
2.7 Alkyl Hydroperoxide Reductase Protein, AhpC (PG0618)
[0119] The most substantial change in P. gingivalis protein
abundance during the transition from haem-excess to haem-limitation
was observed with an alkyl hydroperoxide reductase protein, AhpC
(PG0618, Table 2) which is a peroxide-scavenging enzyme that has
been shown to play an important role in peroxide resistance in P.
gingivalis.sup.(65). In P. gingivalis, formation of a layer of the
.mu.-oxo bis-haem form of iron PPIX with oxygen on the cell surface
is thought to act as an oxidative buffer due to its inherent
catalase-like activity.sup.(11). This layer may also serve as a
cell surface storage of iron and PPIX.sup.(70). During
haem-limitation depletion of this source of iron PPIX was shown to
result in an increased susceptibility to oxidative stress.sup.(64).
The substantial increase in abundance of alkyl hydroperoxide
reductase during haem-limitation could therefore be in response to
the increased oxidative stress caused by the absence/reduction of
the .mu.-oxo bishaem layer. OxyR an oxygen sensitive
transcriptional activator also plays a role in the expression of
alkyl hydroperoxide during anaerobic growth.sup.(71) where a P.
gingivalis OxyR.sup.- mutant shows decrease of 16 fold in gene
expression. More recently Duran-Pinedo and co-workers also
demonstrated the positive regulation of aphC expression by the RprY
response regulator. The substantial increase in abundance thus
suggests haem availability may have a role in RprY and
OxyR-controlled gene expression. Interestingly the very high Codon
Adaption Index (CAI) value of this protein (0.838) suggests this
protein is able to be highly expressed in the cell for rapid
induction in response to such stress.
[0120] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0121] All publications mentioned in this specification are herein
incorporated by reference. Any discussion of documents, acts,
materials, devices, articles or the like which has been included in
the present specification is solely for the purpose of providing a
context for the present invention. It is not to be taken as an
admission that any or all of these matters form part of the prior
art base or were common general knowledge in the field relevant to
the present invention as it existed anywhere before the priority
date of each claim of this application.
[0122] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
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Sequence CWU 1
1
371484PRTPorphyromonas gingivalis (PG0350) 1Met Lys Arg Lys Pro Leu
Phe Ser Ala Leu Val Ile Leu Ser Gly Phe1 5 10 15Phe Gly Ser Val His
Pro Ala Ser Ala Gln Lys Val Pro Ala Pro Val 20 25 30Asp Gly Glu Arg
Ile Ile Met Glu Leu Ser Glu Ala Asp Val Glu Cys 35 40 45Thr Ile Lys
Ile Glu Ala Glu Asp Gly Tyr Ala Asn Asp Ile Trp Ala 50 55 60Asp Leu
Asn Gly Asn Gly Lys Tyr Asp Ser Gly Glu Arg Leu Asp Ser65 70 75
80Gly Glu Phe Arg Asp Val Glu Phe Arg Gln Thr Lys Ala Ile Val Tyr
85 90 95Gly Lys Met Ala Lys Phe Leu Phe Arg Gly Ser Ser Ala Gly Asp
Tyr 100 105 110Gly Ala Thr Phe Ile Asp Ile Ser Asn Cys Thr Gly Leu
Thr Ala Phe 115 120 125Asp Cys Phe Ala Asn Leu Leu Thr Glu Leu Asp
Leu Ser Lys Ala Asn 130 135 140Gly Leu Thr Phe Val Asn Cys Gly Lys
Asn Gln Leu Thr Lys Leu Asp145 150 155 160Leu Pro Ala Asn Ala Asp
Ile Glu Thr Leu Asn Cys Ser Lys Asn Lys 165 170 175Ile Thr Ser Leu
Asn Leu Ser Thr Tyr Thr Lys Leu Lys Glu Leu Tyr 180 185 190Val Gly
Asp Asn Gly Leu Thr Ala Leu Asp Leu Ser Ala Asn Thr Leu 195 200
205Leu Glu Glu Leu Val Tyr Ser Asn Asn Glu Val Thr Thr Ile Asn Leu
210 215 220Ser Ala Asn Thr Asn Leu Lys Ser Leu Tyr Cys Ile Asn Asn
Lys Met225 230 235 240Thr Gly Leu Asp Val Ala Ala Asn Lys Glu Leu
Lys Ile Leu His Cys 245 250 255Asn Asn Asn Gln Leu Thr Ala Leu Asn
Leu Ser Ala Asn Thr Lys Leu 260 265 270Thr Thr Leu Ser Phe Phe Asn
Asn Glu Leu Thr Asn Ile Asp Leu Ser 275 280 285Asp Asn Thr Ala Leu
Glu Trp Leu Phe Cys Asn Gly Asn Lys Leu Thr 290 295 300Lys Leu Asp
Val Ser Ala Asn Ala Asn Leu Ile Ala Leu Gln Cys Ser305 310 315
320Asn Asn Gln Leu Thr Ala Leu Asp Leu Ser Lys Thr Pro Lys Leu Thr
325 330 335Thr Leu Asn Cys Tyr Ser Asn Arg Ile Lys Asp Thr Ala Met
Arg Ala 340 345 350Leu Ile Glu Ser Leu Pro Thr Ile Thr Glu Gly Glu
Gly Arg Phe Val 355 360 365Pro Tyr Asn Asp Asp Glu Gly Gly Glu Glu
Glu Asn Val Cys Thr Thr 370 375 380Glu His Val Glu Met Ala Lys Ala
Lys Asn Trp Lys Val Leu Thr Ser385 390 395 400Trp Gly Glu Pro Phe
Pro Gly Ile Thr Ala Leu Ile Ser Ile Glu Gly 405 410 415Glu Ser Glu
Tyr Ser Val Tyr Ala Gln Asp Gly Ile Leu Tyr Leu Ser 420 425 430Gly
Met Glu Gln Gly Leu Pro Val Gln Val Tyr Thr Val Gly Gly Ser 435 440
445Met Met Tyr Ser Ser Val Ala Ser Gly Ser Ala Met Glu Ile Gln Leu
450 455 460Pro Arg Gly Ala Ala Tyr Val Val Arg Ile Gly Ser His Ala
Ile Lys465 470 475 480Thr Ala Met Pro 2428PRTPorphyromonas
gingivalis (PG1374) 2Met Lys Leu Ser Ser Lys Lys Ile Leu Ala Ile
Ile Ala Leu Leu Thr1 5 10 15Met Gly His Ala Val Gln Ala Gln Phe Val
Pro Ala Pro Thr Thr Gly 20 25 30Ile Arg Met Ser Val Thr Thr Thr Lys
Ala Val Gly Glu Lys Ile Glu 35 40 45Leu Leu Val His Ser Ile Glu Lys
Lys Gly Ile Trp Ile Asp Leu Asn 50 55 60Gly Asp Ala Thr Tyr Gln Gln
Gly Glu Glu Ile Thr Val Phe Asp Glu65 70 75 80Ala Tyr His Glu Tyr
Thr Ile Gly Thr Gln Thr Leu Thr Ile Tyr Gly 85 90 95Asn Thr Thr Arg
Leu Gly Cys Arg Ser Thr Gly Ala Thr Ala Val Asp 100 105 110Val Thr
Lys Asn Pro Asn Leu Thr Tyr Leu Ala Cys Pro Lys Asn Asn 115 120
125Leu Lys Ser Leu Asp Leu Thr Gln Asn Pro Lys Leu Leu Arg Val Trp
130 135 140Cys Asp Ser Asn Glu Ile Glu Ser Leu Asp Leu Ser Gly Asn
Pro Ala145 150 155 160Leu Ile Ile Leu Gly Cys Asp Arg Asn Lys Leu
Thr Glu Leu Lys Thr 165 170 175Asp Asn Asn Pro Lys Leu Ala Ser Leu
Trp Cys Ser Asp Asn Asn Leu 180 185 190Thr Glu Leu Glu Leu Ser Ala
Asn Pro Arg Leu Asn Asp Leu Trp Cys 195 200 205Phe Gly Asn Arg Ile
Thr Lys Leu Asp Leu Ser Ala Asn Pro Leu Leu 210 215 220Val Thr Leu
Trp Cys Ser Asp Asn Glu Leu Ser Thr Leu Asp Leu Ser225 230 235
240Lys Asn Ser Asp Val Ala Tyr Leu Trp Cys Ser Ser Asn Lys Leu Thr
245 250 255Ser Leu Asn Leu Ser Gly Val Lys Gly Leu Ser Val Leu Val
Cys His 260 265 270Ser Asn Gln Ile Ala Gly Glu Glu Met Thr Lys Val
Val Asn Ala Leu 275 280 285Pro Thr Leu Ser Pro Gly Ala Gly Ala Gln
Ser Lys Phe Val Val Val 290 295 300Asp Leu Lys Asp Thr Asp Glu Lys
Asn Ile Cys Thr Val Lys Asp Val305 310 315 320Glu Lys Ala Lys Ser
Lys Asn Trp Arg Val Phe Asp Phe Asn Gly Asp 325 330 335Ser Asp Asn
Met Leu Pro Tyr Glu Gly Ser Pro Thr Ser Asn Leu Ala 340 345 350Val
Asp Ala Pro Thr Val Arg Ile Tyr Pro Asn Pro Val Gly Arg Tyr 355 360
365Ala Leu Val Glu Ile Pro Glu Ser Leu Leu Gly Gln Glu Ala Ala Leu
370 375 380Tyr Asp Met Asn Gly Val Lys Val Tyr Ser Phe Ala Val Glu
Ser Leu385 390 395 400Arg Gln Asn Ile Asp Leu Thr His Leu Pro Asp
Gly Thr Tyr Phe Phe 405 410 415Arg Leu Asp Asn Tyr Thr Thr Lys Leu
Ile Lys Gln 420 4253393PRTPorphyromonas gingivalis (PG1019) 3Met
Lys Lys Asn Phe Leu Phe Phe Ser Leu Val Leu Ala Ala Ile Met1 5 10
15Ser Leu Leu Ser Ser Cys Ala Lys Asp Thr Pro Asp Ala Pro Glu Gln
20 25 30Tyr Ala Ile Thr Ile Arg Ala Lys Leu Pro Glu Gly Ser Thr Ile
Glu 35 40 45Ser Leu Ala Gly Ile Ala Ile Glu Phe Leu Asp Leu Arg Thr
Gln Gln 50 55 60Lys Val Glu Lys Gln Leu Asp Lys Ala Gly Val Cys Ser
Leu Ser Leu65 70 75 80Asp Ala Ser Val Tyr Thr Ile Thr Ile Arg Gly
Glu Ile Gly Asn Asn 85 90 95Ser Ile Val Ala Ile Lys Glu Asn Tyr Ser
Ile Ala Glu Asn Thr Thr 100 105 110Leu Glu Leu Pro Leu Ile Val Thr
Lys Ile Arg Pro Ser Gly Leu Leu 115 120 125Phe Lys Glu Val Phe Phe
Asn Gly Glu Thr Asn Asn Gly Gln Met Met 130 135 140His Pro Asp Gln
Tyr Phe Val Ile Tyr Asn Asn Ser Asp Lys Val Val145 150 155 160Tyr
Ala Asp Gly Val Ala Phe Gly Leu Ala Ala His Ala Asn Val Thr 165 170
175Gly Glu Asp Ala Phe Thr Glu Glu Leu Thr Lys Asn Asn Arg Ile Val
180 185 190Leu Ser Met Ile Tyr Thr Ile Pro Gly Asn Gly Ser Gln Tyr
Pro Ile 195 200 205Gln Pro Gly Gly Gln Leu Val Ile Ala Gly Thr Ala
Ile Asn His His 210 215 220Asp Ala Glu His Pro Asn Ser Val Asp Leu
Ser Gly Ala Asp Leu Glu225 230 235 240Val Tyr Glu Pro Asp Gln Pro
Ala Asn Phe Gly Gln Asp Val Asp Asn 245 250 255Pro Asn Val Pro Asn
Met Val Lys Ile Phe Asn Arg Phe Gly Val Phe 260 265 270Met Met His
Pro Arg Gly Phe Ile Pro Pro Val Leu Phe Glu Ile Asp 275 280 285Glu
Pro Ile Glu Thr Phe Leu Ala Lys Asn Gln Phe Glu Tyr Thr Asn 290 295
300Asn Asp Gly Glu Asn Ile Met Leu Tyr Ala Val Pro Val Glu Asn
Val305 310 315 320Leu Asp Gly Ile Glu Thr Ala Asn Thr Gly Asn Met
Lys Val Lys Ser 325 330 335Leu Pro Val Thr Val Asp Lys Ser Met Ile
Gly Val Pro Gly Cys His 340 345 350Arg Gly Ile Leu Ile Leu Arg Lys
Thr Glu Glu Lys Asn Gly Arg Thr 355 360 365Tyr Met Ile Asp Thr Asn
Asp Ser Glu Asn Asp Cys Ile Ala Arg Gln 370 375 380Gly Gln Asn Ser
Phe Pro Ala Arg Phe385 3904188PRTPorphyromonas gingivalis (PG0618)
4Met Thr Pro Ile Leu Asn Thr Val Phe Pro Glu Phe Lys Leu Asn Ala1 5
10 15Tyr His Asn Gly Glu Phe Lys Val Ile Thr Asn Glu Asp Leu Lys
Gly 20 25 30Lys Trp Ser Leu Val Val Phe Tyr Pro Gly Asp Phe Thr Phe
Val Cys 35 40 45Pro Thr Glu Leu Glu Asp Leu Ala Asn Lys Tyr Glu Glu
Phe Lys Gln 50 55 60Leu Gly Val Glu Val Tyr Ser Cys Ser Cys Asp Thr
His Phe Val His65 70 75 80Lys Ala Trp Ala Asp Ala Ser Pro Ala Ile
Lys Lys Val Gln Tyr Pro 85 90 95Met Leu Ala Asp Pro Ser Gly Ala Leu
Thr Arg Asp Leu Gly Ile Leu 100 105 110Ile Asp Asp Val His Met Ala
Tyr Arg Gly Ser Phe Val Ile Asn Pro 115 120 125Glu Gly Ile Ile Lys
Ile Val Glu Leu Asn Asp Asn Ser Val Gly Arg 130 135 140Asp Ala Glu
Glu Ile Leu Arg Lys Ile Lys Ala Ala Gln Tyr Val Ala145 150 155
160Ala His Asp Gly Gln Val Cys Pro Ala Lys Trp Arg Glu Gly Gln Gln
165 170 175Thr Leu Lys Pro Ser Ile Asp Leu Val Gly Lys Ile 180
18551455DNAPorphyromonas gingivalis (PG0350) 5atgaaaagaa aaccgctatt
ctcagccctt gtaatccttt ccggcttctt cggatcggtt 60cacccggcct cagcacagaa
agttcctgca cccgtcgatg gcgagcgcat tatcatggag 120ctaagtgaag
ccgatgtgga gtgtacaatc aaaatagaag ccgaggatgg ctatgccaac
180gacatttggg cagacctcaa cggaaacggc aagtacgatt cgggggagag
gctcgattca 240ggtgagtttc gtgatgttga gttcagacaa acaaaggcca
tcgtctatgg caaaatggcc 300aaattcttgt ttagaggttc ttctgcaggg
gactatggtg ctacctttat agatattagc 360aattgtaccg gcctgactgc
tttcgactgc tttgccaatc tgctgacaga actcgatctg 420tccaaagcaa
acggtctgac ttttgtaaac tgcggcaaaa accagctgac caagcttgac
480ctgcccgcaa atgcggacat tgagacgctg aactgctcca aaaacaagat
aacgagtctc 540aacctatcga cctataccaa gctgaaagag ctttatgtgg
gcgacaacgg gctgacagcc 600ttggatctct ccgccaatac gctcctcgaa
gagctggtgt attctaacaa cgaggtgact 660acgataaacc tgtctgccaa
tacgaacttg aaaagcctgt attgcataaa caataagatg 720accggactcg
atgtcgcagc caacaaagag ctgaaaatac tccactgcaa caacaatcag
780ctgaccgccc tcaatctctc ggccaatacc aagctgacga ctctaagctt
cttcaacaac 840gagctgacaa atatcgatct ctccgacaac acggctttgg
agtggctttt ctgcaacggc 900aataagctga cgaagttaga tgtatctgcc
aacgccaatc tgatagcact gcaatgcagc 960aacaaccagc tgactgctct
ggatctgtca aaaacgccga aactgacaac gttgaattgc 1020tactccaacc
ggatcaaaga taccgccatg cgtgcattga tcgaaagcct gcctacgatc
1080actgaaggag aaggcaggtt cgttccttac aacgacgatg aaggaggaga
agaggagaac 1140gtgtgtacaa ccgaacacgt ggaaatggcc aaggccaaga
attggaaggt acttacctcg 1200tggggagagc ctttccccgg aataacggct
ttgatttcca tcgaaggtga gagcgaatat 1260tccgtatatg ctcaagatgg
catcctctac ctctccggta tggagcaggg cttgcccgtt 1320caggtatata
ccgtgggagg aagcatgatg tactcatctg tcgcttccgg atcagccatg
1380gaaatacagc tcccgagagg tgcagcctat gtagtacgta tcggcagcca
tgcgatcaaa 1440accgcgatgc cgtaa 145561287DNAPorphyromonas
gingivalis (PG1374) 6atgaaacttt catctaagaa aatcttagca atcattgcat
tgctgacgat gggacatgct 60gtgcaggcac agtttgttcc ggctcccacc acagggattc
gcatgtctgt cactacaacc 120aaggccgtag gcgaaaaaat cgaattgttg
gttcattcca tagagaagaa aggcatctgg 180atcgatctca atggggatgc
cacttaccaa caaggagagg aaataaccgt attcgatgag 240gcataccacg
aatacacgat cgggacgcaa accctcacta tctatggtaa tacgacccga
300ttgggctgtc gatctaccgg tgcaacggct gtcgatgtaa cgaaaaaccc
taatctgacc 360tatctcgcat gcccgaaaaa taatctgaaa tcattggact
tgacgcaaaa cccaaagctg 420ctgcgagttt ggtgcgactc taacgaaata
gaaagtttgg acctgagtgg caatccggct 480ttgatcatcc tcggctgtga
caggaataag ctgactgagc tgaagaccga taacaacccc 540aagttggcct
ctctttggtg ttctgataat aacctgacgg agttggaact cagtgccaat
600cctcgtctca atgatctttg gtgcttcggt aatcggatca cgaaactcga
tctgagtgcc 660aatcctctat tggtaacact ttggtgcagt gacaatgagc
tttcgacctt ggatctttcc 720aagaattcgg acgttgctta cctttggtgc
tcatcgaaca aacttacatc cttgaatctg 780tcgggggtga agggactgag
tgttttggtt tgtcattcca atcagatcgc aggtgaagaa 840atgacgaaag
tggtgaatgc tttgcccaca ctatctcccg gcgcaggcgc tcagagcaag
900ttcgtcgttg tagacctcaa ggacactgat gagaagaata tctgtaccgt
aaaggatgtg 960gaaaaagcta aaagtaagaa ctggcgagta tttgacttca
acggtgattc tgacaatatg 1020cttccatacg aaggaagtcc gacatcgaac
ttggcagtag atgctcccac tgtcaggata 1080tatcccaatc cggtaggaag
atatgcgctc gtcgagatcc ccgagtctct tttagggcag 1140gaagctgctt
tatacgatat gaatggggta aaagtctata gtttcgcggt agagtctctt
1200cgtcagaaca ttgacctgac acatcttccc gacggcactt atttcttccg
tctcgataac 1260tataccacta agctcatcaa acagtag
128771182DNAPorphyromonas gingivalis (PG1019) 7atgaaaaaaa
attttctttt tttctccctc gttttagcag ccatcatgtc gttgctgtca 60tcttgtgcca
aggatacgcc ggatgcgccc gaacagtacg ctatcactat ccgtgccaaa
120ctaccggaag gcagtacgat agagagtctc gcaggtatag ccatcgaatt
cctcgatctt 180cgtacccaac agaaagtgga aaaacagctc gacaaagccg
gtgtttgctc tcttagtctg 240gatgccagtg tatatacgat tacgatacgt
ggcgaaatag ggaacaacag tatcgttgcc 300atcaaggaaa actattccat
cgcagagaat actaccttgg agcttccact cattgtgacg 360aagatccgcc
cttccggtct gctgttcaaa gaagtatttt tcaatggaga gaccaacaac
420gggcagatga tgcacccgga tcagtacttc gtcatataca ataatagcga
taaggtggtc 480tatgccgatg gtgtcgcttt cggtcttgcc gcacatgcca
acgtaacagg tgaagacgct 540ttcaccgagg agttgaccaa gaacaaccgc
atcgtccttt ccatgatcta taccattccc 600ggcaacggtt cgcagtatcc
catccaaccc ggtggtcagc tcgtgatagc cggaacggcc 660atcaatcacc
acgatgccga gcatccgaat tccgtggact tgagcggtgc cgatttggaa
720gtctatgagc cggatcagcc cgcaaacttc ggacaggatg tggacaaccc
caatgttccc 780aatatggtga agatatttaa tcgattcggt gttttcatga
tgcatcccag aggatttatc 840cctcctgttc ttttcgagat agatgagccg
atcgagactt tcctggccaa gaaccagttc 900gagtacacga acaatgatgg
agagaacatt atgctctatg ccgttccggt ggaaaatgtg 960ttggatggta
tcgagaccgc caataccggt aatatgaaag tcaagtccct gcccgtgaca
1020gtggataagt ccatgatcgg tgtaccgggt tgccaccgtg gcatactcat
tcttcgcaag 1080acggaagaaa agaatggccg tacctatatg atcgacacca
acgactctga aaatgactgt 1140atagcccgtc aaggacaaaa ctcttttcct
gcaagattct aa 11828567DNAPorphyromonas gingivalis (PG0618)
8atgactccta tcctgaacac cgttttcccc gagttcaaac tcaatgccta tcacaatggc
60gaattcaaag taatcaccaa cgaagacttg aaaggcaagt ggtctttggt cgttttctat
120cccggtgact ttacctttgt atgcccgacg gaattggaag acctggccaa
taaatatgaa 180gaattcaagc aacttggagt agaggtttac tcttgcagtt
gcgataccca cttcgtacac 240aaggcttggg ccgacgcttc tcctgctatc
aagaaggtac agtatcccat gttggccgat 300ccctccggtg cactcactcg
cgatctgggt atcctgatcg atgatgttca tatggcttac 360cgtggctctt
tcgtgattaa ccccgaaggc attatcaaaa tcgtagagct gaacgacaac
420agcgtaggcc gtgatgcaga agagatcctc cgtaagatca aggctgcaca
atacgtagct 480gctcacgatg gtcaggtatg tccggccaag tggcgtgaag
gtcagcagac actgaaaccg 540agcattgatc tcgttggtaa gatctaa
567910PRTPorphyromonas gingivalis 9Leu Val Asp Leu Asn Cys Phe Asp
Ile Lys1 5 101028DNAArtificial SequencePrimer 10agagggccct
agcaatcatt gcattgct 281128DNAArtificial SequencePrimer 11tgcgacgtcg
tgttaccaat agaggatt 281228DNAArtificial SequencePrimer 12tgactgcagg
ctttcgacct tggatctt 281328DNAArtificial SequencePrimer 13tcgcatatga
agaaataagt gccgtcgg 281412PRTPorphyromonas gingivalis 14Gly Gln Asp
Glu Met Asn Glu Ile Leu Cys Glu Lys1 5 10159PRTPorphyromonas
gingivalis 15Cys Tyr Asp Pro Gly Val Thr Pro Lys1
51616PRTPorphyromonas gingivalis 16Asp Ala Gly Met Ser Ala Gln Ser
His Glu Tyr Cys Val Glu Val Lys1 5 10 151725PRTPorphyromonas
gingivalis 17Glu Gly Leu Thr Ala Thr Thr Phe Glu Glu Asp Gly Val
Ala Ala Gly1 5 10 15Asn His Glu Tyr Cys Val Glu Val Lys 20
251815PRTPorphyromonas gingivalis 18Cys Val Asn Val Thr Val Asn Ser
Thr Gln Phe Asn Pro Val Lys1 5 10 151911PRTPorphyromonas gingivalis
19Cys Gln
Ile Leu Ile Glu Asn His Asp Lys Arg1 5 102012PRTPorphyromonas
gingivalis 20Tyr Pro Ser Leu Cys Thr Thr Ser Val Ile Gly Lys1 5
102115PRTPorphyromonas gingivalis 21Cys Val Val Asn Ser Pro Gly Gly
Gln Thr Ala Ser Met Ala Lys1 5 10 152213PRTPorphyromonas gingivalis
22Phe Ser Asn Leu Pro Val Leu Gly Gly Glu Ser Cys Arg1 5
102325PRTPorphyromonas gingivalis 23Phe Val Pro Tyr Asn Asp Asp Glu
Gly Gly Glu Glu Glu Asn Val Cys1 5 10 15Thr Thr Glu His Val Glu Met
Ala Lys 20 252415PRTPorphyromonas gingivalis 24Ile Ile Met Glu Leu
Ser Glu Ala Asp Val Glu Cys Thr Ile Lys1 5 10
152520PRTPorphyromonas gingivalis 25Ile Leu His Cys Asn Asn Asn Gln
Leu Thr Ala Leu Asn Ser Leu Ser1 5 10 15Ala Asn Thr Lys
202616PRTPorphyromonas gingivalis 26Leu Asp Leu Pro Ala Asn Ala Asp
Ile Glu Thr Leu Asn Cys Ser Lys1 5 10 152719PRTPorphyromonas
gingivalis 27Gly Leu Ser Val Leu Val Cys His Ser Asn Gln Ile Ala
Gly Glu Glu1 5 10 15Met Thr Lys2811PRTPorphyromonas gingivalis
28Asn Pro Asn Leu Thr Tyr Leu Ala Cys Pro Lys1 5
102912PRTPorphyromonas gingivalis 29Met Glu Thr Glu Leu Ala Gln Ile
Cys Tyr Ser Lys1 5 103017PRTPorphyromonas gingivalis 30Tyr Asp Ala
Ser Asn Glu Leu Arg Pro Thr Ile Leu Cys Ile Tyr Gly1 5 10
15Lys3119PRTPorphyromonas gingivalis 31Met Asn Ser Asp Glu Leu Phe
Glu Glu Ile Thr Tyr Pro Gly Tyr Thr1 5 10 15Ile Cys
Arg3216PRTPorphyromonas gingivalis 32Thr Tyr Met Ile Asp Thr Asn
Asp Ser Glu Asn Asp Cys Ile Ala Arg1 5 10 153317PRTPorphyromonas
gingivalis 33Phe Gly Ser Val Leu Glu Val Phe Gln Gln Val Tyr Glu
His Glu Cys1 5 10 15Lys3413PRTPorphyromonas gingivalis 34Glu Glu
His Glu Leu Val Cys Ala Ala Ser Thr Leu Lys1 5
103516PRTPorphyromonas gingivalis 35Ala Ala Gln Tyr Val Ala Ala His
Asp Gly Gln Val Cys Pro Ala Lys1 5 10 153618PRTPorphyromonas
gingivalis 36Arg Pro Val Ser Cys Pro Glu Cys Pro Glu Pro Thr Gln
Pro Thr Val1 5 10 15Thr Arg3716PRTPorphyromonas gingivalis 37Arg
Pro Val Ser Cys Pro Glu Cys Pro Glu Val Thr Pro Val Thr Lys1 5 10
15
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References